Manufacture of healthy snack bars supplemented with moringa sprout powder

LWT - Food Science and Technology 154 (2022) 112828 Available online 18 November 2021 0023-6438/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Manufacture of healthy snack bars supplemented with moringa sprout powder Karín E. Coello a , Juana Frias b , Cristina Martínez-Villaluenga b , María Elena Cartea c , Pablo Velasco c , Elena Pe˜ nas b , * a Polytechnic University, Escuela Superior Polit´ ecnica del Litoral, ESPOL, Faculty of Mechanical Engineering and Production Sciences, Food Engineering Program, Campus Gustavo Galindo Km 30.5 Vía Perimetral, P.O. Box 09-01-5863, Guayaquil, Ecuador b Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Juan de la Cierva 3, Madrid, 28006, Spain c Group of Genetics, Breeding and Biochemistry of Brassicas, Mision Biologica de Galicia (MBG-CSIC), 36080, Pontevedra, Spain ARTICLE INFO Keywords: Moringa Sprouting Snack bar Health benefits Nutritional value ABSTRACT The present study focuses on the utilization of moringa sprout powder (MSP) for development of healthy and ready-to-eat snack bars. Different MSP proportions (9–26%) were evaluated for snack bar formulation and compared with control formulation (0% MSP). 18% MSP-containing prototype (18MSP) was selected as the optimal formulation based on the results of the fracturability assay. 18MSP bar exhibited higher protein, ash and fat and lower total carbohydrate contents than control, and was a good source of B 1 and B 2 vitamins (983.0 and 94.1 μ g/100 g dry matter, dm, respectively) and minerals. 18% MSP inclusion significantly (p ≤0.05) enhanced the contents of total amino acid (80.8 mg/g dm), the relative content of unsaturated fatty acids (FA) (64.0 g/100 g of total FA), γ-aminobutyric acid (GABA, 43.3 mg/100 g dm), and total and individual glucosinolates (GLS) (29.6, 1.1, 0.21 and 30.9 μ mol/g dm for glucomoringin, glucosinalbin, glucotropaeolin and total GLS, respectively), as well as the antioxidant activity (647.2 mg TE/100 g dm). 18MSP had good sensorial acceptability, mainly due to its appearance, texture and odour. Questionnaires performed to potential Ecuadorian consumers indicated the favourable intention of purchasing the novel MSP-supplemented snack bar. 1. Introduction In recent years, changes undergone in people lifestyles, the lack of time to prepare a proper meal and the constant information regarding the influence of diet on health status have drastically altered consumer eating patterns. Snacks are ready-to-eat palatable and affordable foods that meet the current consumer demands regarding foods capable of being portable and consumed quickly (Mattes, 2018). Snacks are one of the fastest growing food market sectors and their consumption has substantially increased in last years in all age groups worldwide (Vatanparast et al., 2020). However, consumption of unhealthy energy-dense snacks constitutes an important risk factor for obesity and cardiovascular disease development (Amrein, Scholz, & Inauen, 2021). Manufacturing healthy snacks with good sensory properties represents a food industry priority in order to improve the well-being, nutritional and health status of consumers while preventing the development of non-communicable diseases (NCD). Cereal-based bars are attractive foods to achieve these purposes since they can be easily consumed between meals to satisfy momentarily hunger and they can be produced from different plant-based sources providing nutritional and health benefits. Moringa oleifera Lam. is a foliaged tree native of northern India that is widely cultivated in tropical/subtropical regions of Africa, Central/ South America and Asia (Leone et al., 2015). All parts of moringa tree are edible and have been used as food as well as in ayurvedic medicine and cosmetic industry since ancient times. Although leaves are the part most frequently used for nutritional purposes, moringa seeds are also valuable raw materials for food applications since they are a good source of proteins, unsaturated FA and minerals (García Milla, Pe˜ nalver, & Nieto, 2021; Singh et al., 2020). Recent studies have shown that moringa seed powder improves the nutritional value of several foodstuffs such as bread, cookies, cakes, snacks and cereal gruels (Aluko, Brai, & Adelore, 2013; Chinma, Gbadamosi, Ogunsina, Oloyede, & Salami, 2014; Jude-Ojei, Lola, Ajayi, & Seun, 2017; Ogunsina, Radha, & Indrani., 2011; Rabie, Ibrahim, Youssif, & El-Ragal, 2020). In the past few years, different technological approaches including * Corresponding author. E-mail address: [email protected] (E. Pe˜ nas). Contents lists available at ScienceDirect LWT journal homepage: www.elsevier.com/locate/lwt https://doi.org/10.1016/j.lwt.2021.112828 Received 23 September 2021; Received in revised form 11 November 2021; Accepted 15 November 2021 LWT 154 (2022) 112828 2 germination have been applied to improve the nutritional quality and health benefits of seeds. During germination, hydrolytic enzymes are activated or de novo synthesized to breakdown stored nutrients in order to fuel seedling growth, leading to numerous physiological/biomolecular changes in seeds (Lemmens et al., 2019). These modifications include degradation of complex carbohydrates, lipids and storage proteins, as well as the synthesis of micronutrients, proteins and fiber. Germination also increases the content of secondary metabolites such as phenolic compounds with recognized antioxidant and anti-inflammatory activity. Similarly, GABA is synthesized during grain germination (Aparicio-García, Martínez-Villaluenga, Frias, & Pe˜ nas, 2021), compound that exhibits various health benefits including reduction of blood pressure, inhibition of carcinogenic cell proliferation, antioxidant and immunomodulatory activities (Sahab, Subroto, Balia, & Utama., 2020). GLS, sulfur-containing secondary metabolites present in Brasicaceae vegetables, are broke down during germination (Coello et al., 2020) to yield a variety of bioactive compounds with recognized antioxidant and anti-carcinogenic activities (Maina, Misinzo, Bakari, & Kim, 2020). Despite germination benefits, its impact on moringa seed quality has been scarcely investigated. A recent study of our group optimized germination parameters to produce a novel moringa sprout powdered (MSP) ingredient enriched in nutrients, GABA and GLS (Coello et al., 2020). Taking into account the recent trends towards the development of functional foods as a mean of preventing the development of NCD, the aim of the present study was to explore the application of MSP to develop innovative and healthy cereal-based snack bars enriched in nutrients and bioactive compounds. 2. Materials and methods 2.1. Chemical, reagents and standards All chemicals used were provided by Sigma-Aldrich (Madrid, Spain) unless otherwise specified. 2.2. Production of moringa sprout powder (MSP) Seeds obtained from moringa trees grown in the Eastern part of Ecuador were germinated at 36 ◦C for 96 h in darkness in a thermostatically-controlled germination cabinet (G-120 model, ASL Snijders International S. L., Tilburg, The Netherlands), according to Aparicio-García et al., (2021). An humidity higher than 90% was maintained during the whole germination process due to the water circulating system present in the germination chamber. These conditions were selected because they maximized the GABA content in moringa (Coello et al., 2020). Sprouts were dried (50 ◦C, 24 h) in a drying cabinet (model KBF 240, Binder, Tuttlingen, Germany), milled using a coffee grinder (Moulinex, France) and passed through a sieve (0.2 mm of diameter) to obtain MSP, that was stored in plastic bags under vacuum at −20 ◦C for further use. 2.3. Manufacturing of snack bars 2.3.1. Ingredients Oat grains and flaked oatmeal were provided by Basesur and Avenas del Pacífico (Guayaquil, Ecuador), respectively, crispy rice was supplied by DULCENAC (Guayaquil, Ecuador), dehydrated coconut was provided by Confites Magus’s (Quito, Ecuador), dehydrated banana slices were supplied by P.E.B.S.A-Productos Elaborados Bolívar (Guayaquil, Ecuador), quinoa seeds were provided by Coprobich (Chimborazo, Ecuador) and panela was supplied by Sugarmill Industry San Carlos (Guayaquil-Ecuador). 2.3.2. Preparation of snack bars Snack bar formulations with different ratio of solid/liquid phases (65/35, w/v and 75/25, w/v) were produced. Liquid phase consisted of a saturated solution of heated panela (75 ◦Brix) acting as a binder syrup. Three snack bars corresponding to each formulation containing different MSP proportions (9–26%) were prepared. Snack bars produced without MSP addition were considered as control samples. Snack bar formulations were coded as control, 6MSP, 9MSP, 12MSP, 18MSP, 19MSP and 26MSP, according to the amount of MSP included: 0%, 6%, 9%, 12%, 18% and 19%, respectively (Table 1). The solid ingredients including MSP were weighted and thoroughly manually mixed with the heated panela solution to obtain a uniform dough structure. Doughs were placed into stainless-steel containers (3.0 x 4.5 ×7.5 cm) and baked (105 ◦C, 15 min) in order to obtain bars with a water activity lower than 0.78, as previously reported (Serna-Cock, Angulo-L´ opez, & Ayala-Aponye, 2015). Bars were prepared in the Laboratory of Development and Food Processing of ESPOL (Guayaquil, Ecuador). Each bar of approximately 35 g was packed in tri-laminated polyethylene-polyethylene-aluminum foil and stored at ambient temperature until further analysis. Fig. S1 (supplementary material) illustrates the different snack bar formulations produced. 2.3.3. Descriptive fracturability analysis The fracturability of snack bars was evaluated in order to select the formulation with the best texture. This analysis was carried out by 10 trained panelists recruited from the staff of Wipala Healthy Snacks (Guayaquil, Ecuador), one of the main snack producers in Ecuador. Control and MSP-supplemented bars were placed in the mouth, compressed between molar teeth, and bit down evenly until bars shattered (Sato et al., 2018). A 5-point non-structured scale was used, where 1 and 5 represent the lowest and the highest cutting force, respectively. 2.4. Proximate composition of snack bars AOAC methods were used to determine protein, fat, ash and total dietary fiber contents. Total carbohydrates (including fiber content) were estimated by difference: 100-(% proteins +% fat +% ash +% water) (Caceres, Martinez-Villaluenga, Amigo, & Frias, 2014). A factor of 5.83 was used for protein content conversion (Coello et al., 2021). The results were expressed in g/100 g on dry matter basis (dm). 2.5. Contents of thiamine and riboflavin Thiamine and riboflavin were extracted after acid hydrolysis of snack bars in an autoclave (15 min, 120 ◦C) followed by enzymatic dephosphorylation with Taka-Diastase (3 h, 45 ◦C), and quantified separately by reverse-phase high performance liquid chromatography (HPLC) (Coello et al., 2020). Results were expressed in μ g/100 g dm. 2.6. Mineral composition Mineral profile was determined by atomic absorption using a Analytikjena ContrAA 700 high-resolution atomic absorption spectrometer (Analytik Jena AG, Jena, Germany) equipped with a Xenon shortarc lamp (GLE, Berlin, Germany) operating in a “hot spot” mode as the radiation source (Alvarez, Fuentes, Guerrero, & Canet, 2017). Results were expressed as mg/kg dm. 2.7. Fatty acid composition FA profile of snack bars was determined by measuring the content of fatty acid methyl esters (FAMEs) by gas chromatography (GS) on an Agilent gas chromatograph (7820A model) with a flame ionization detector (FID) (Aparicio-García et al., 2021). A FAME standard mixture (FAME 37 +PUFA N◦2 Animal Source +PUFA N◦3 Menhaden oil) was used for identification of individual FAMEs in snack bars. Results were expressed as mg/g dm. K.E. Coello et al. LWT 154 (2022) 112828 3 2.8. Amino acid profile The content of protein amino acids was determined by acid hydrolysis, derivatization and HPLC quantification (Pe˜ nas, Gomez, Frias, Baeza, & Vidal-Valverde, 2011). The results were expressed as mg/g dm. 2.9. γ-aminobutyric acid content GABA was extracted in distilled water and quantified by HPLC, as previously reported (Caceres et al., 2014). The results were expressed as mg GABA/100 g dm. 2.10. Individual and total GLS Total GLS were extracted from 100 g of snack bars-derived flour following a two-step extraction and desulfatation procedure (Kliebenstein et al., 2001). After freezing (−80 ◦C) and freeze-drying these samples (−0.1 mbar, 24 h, Gamma 2–16 Christ freeze dryer), GLS content in the resultant extracts was quantified by Ultra-High-Performance Liquid Chromatography (UHPLC) using an UPLC Nexera LC-30AD chromatograph (Shimadzu, Kyoto, Japan) equipped with a Nexera SIL-30AC injector and SPD-M20A photodiode array detector (Coello et al., 2021). Results were expressed as μ mol/g dm. 2.11. Content of soluble phenolic compounds (SPC) SPC were extracted from snack bar flours (100 mg) in 80% methanol/0.1% formic (1 mL) acid under continuous agitation (2000 rpm, 20 ◦C, 15 min). Samples were then centrifuged (10,000 rpm, 20 ◦C, 5 min) (Sorval RC 6 Plus centrifuge ThermoFisher, Madrid, Spain). Supernatants were collected, and 1 mL of 70% acetone/0.1% formic acid was added to the pellet for a second extraction in the same conditions. Supernatants from the first and second extractions were combined and the volume was made up to 2 mL with bidistilled water. SPC were quantified in the extract by Fast Blue BB assay (Pico, Pismag, Laudouze, & Martinez, 2020). Results were expressed as mg of gallic acid equivalents (GAE)/100 g dm. 2.12. Oxygen radical absorbance capacity (ORAC) Antioxidant activity was determined in the extracts obtained for SPC quantification by ORAC assay (Caceres et al., 2014). The fluorescence was quantified in a microplate reader (Synergy HT, BioTek Instruments, Winnoski, VT, USA) every minute at λexc 485 nm and λem 520 nm. The results were expressed as mg of Trolox equivalents (TE)/100 g dm. 2.13. Sensory analysis A total of 30 panelists who declared to consume snack bars at least once a week were recruited among the students and employees of ESPOL. Sensory evaluation was carried out in individual boxes under white light. Two snack bars (control and MSP-supplemented bars) were presented to panelists in white plastic plates. After tasting each sample, panelists rinse their mouth with bottled water. A hedonic scale was used to evaluate 5 sensory attributes, including appearance, aroma, taste, texture and overall acceptability, where +3 means “like extremely”, +2 “like moderately”, +1 “like slightly”, 0 “indifference”, −1 “dislike slightly”, −2 “dislike moderately” and −3 “dislike extremely”. It is important to highlight that this was a preliminary sensory test to elucidate whether the novel snack formulated will be accepted by Ecuadorian consumers. 2.14. Estimation of manufacturing process capacity The capacity of producing snack bars at industrial scale was calculated according to the following equation: P η =(mf/mi)(Eq. 1) where P η is the total capacity of production, m i is the initial mass and m f is the final mass resulting. 2.15. Estimation of production costs and selling price The potential costs derived from industrial production of MSPsupplemented snack bars were performed for the production of 25,000 units as described by Guerrero Macías (2013), according to the following equation: TC =M+DH +IC +T(Eq. 2) where TC represents the total cost, M is the cost of the materials, DH is the total direct cost derived from work hand, IC is the indirect manufacturing costs and T corresponds to product taxes. The production cost per unit (PUC) was calculated as follows: PUC =TC/U(Eq. 3) where U represents total product units manufactured per month. The selling price (SP) was calculated as follows: SP =PUC +CM (Eq. 4) where CM is the contribution margin, calculated as follows: CM =0.4∗SPa (Eq.5) where SPa is the average of the prices for commercial snack bars in Ecuador, ranging from 0.36 to 1.14 $. 2.16. Estimation of consumer’s willingness to purchase MSPsupplemented snack bars A questionnaire comprising 10 questions was administered to young adults (20–34 years) of middle social class (income from the basic salary Table 1 Formulations for control and MSP snack bars. SI/LI ratio Snack bar formulation % SI MSP Oat grain Flaked oatmeal Quinoa Crispy rice Dry banana Dry coconut 65/35 Control 0 13 10 13 20 9 0.1 9MSP 9 12 9 8 12 9 6 18MSP 18 10 7 6 11 8 5 26MSP 26 9 6 5 9 7 3 75/25 Control 0 15 13 16 20 11 0.1 6MSP 6 14 9 14 12 10 10 12MSP 12 13 8 13 11 10 9 19MSP 19 12 7 11 10 8 8 SI: Solid ingredients, LI: Liquid ingredients. K.E. Coello et al. LWT 154 (2022) 112828 4 onwards) who attended to supermarkets and shopping centers of Guayaquil (Ecuador) from June 11th, 2018 to June 18th, 2018. A guarantee of anonymity was used to counteract inclinations to offer “socially correct” answers. The questionnaire comprised questions devoted to snack bar consumption frequency, motives influencing snack bar consumption, knowledge of moringa and its nutritional and health benefits, willingness to buy a snack bar supplemented with moringa, and maximum price willing to pay for this novel and healthy product. The eligibility criteria to complete the questionnaire was represented by the answer to the question “Do you consume snack bars?“. In the case of negative response, the questionnaire was not conducted. A total of 104 useable questionnaires were obtained, that were considered a representative sample of the reference population (adult population ranging 20–34 years of Guayaquil, estimated in 592,736 persons, being the total population of 2.291.158 persons) (INEC, 2011). 2.17. Statistical analysis Each snack bar formulation was produced in triplicate and all analyses were performed in duplicate. Results were expressed as the mean ±standard deviation. Experimental data were submitted to one-way analysis of variance (ANOVA) and post hoc Duncan multiple range test (p ≤0.05) using Statgraphics Centurion XII software, version 17.2.07 (Statistical graphics Co., Rockville, MD, USA). 3. Results and discussion 3.1. Selection of the optimal snack bar formulation Since fracturability, defined as the force applied by the molar teeth to crumble or cracked food, is one of the most important attributes of snack bars (Kumar, Mohanty, & Yashaswini., 2018), a preliminary descriptive analysis of fracturability was performed in the different snack bar prototypes in order to select the optimal formulation. No significant differences (p ≤0.05) in fracturability were found between the different snack bar formulations (Table 2). However, it was observed that formulations containing 75/25 solid to liquid ratio exhibited worse consistency than those containing a proportion of 65/35. Taking into account this result, together with the fact that low fracturability value is a desirable property for snack bars and that higher MSP content would improve nutritional and functional quality of bars, we selected 18MSP as the optimal formulation that meet all these features. 3.2. Proximate composition Table 3 details the proximate composition of the selected MSPenriched snack bar formulation (18MSP) compared to control bar (0MSP). It was evident that the addition of MSP significantly (p ≤0.05) increased the content of protein and ash (48%) and fat (more than 4fold) as compared to control bar, while the moisture (8%) and total carbohydrates (15%) contents were reduced. 18MSP moisture content is lower than that reported in other fruitand grain-based snack bars (Momanyi, Owino, & Makokha, 2020; Da Silva, Siqueira, Carvalho do Lago, Rosell, & Barros Vilas Boas, 2014). Low moisture content is a desirable attribute to increase the microbial stability and shelf-life of the product and to obtain snacks with crunchy texture (Momanyi et al., 2020). Germinated moringa flour is a good source of proteins and minerals (Coello et al., 2020; Le´ on-L´ opez et al., 2019), thus explaining the increased protein and ash contents found in 18MSP formulation compared to control. Moringa seed contains high lipids levels (13–46%) (Saa, Formbarng, Ndjantou, & Njintang, 2019), mainly unsaturated FA (Al Juhaimi, Ghafoor, Babiker, Mattha¨ aus, & ¨ Ozcan, 2017), responsible for the higher fat content in 18 MSP compared to control. The replacement of oat, an excellent source of starch, by MSP where the starch is partially hydrolysed to reducing sugars by activation of endogenous α -amylases during germination (Coello et al., 2020), could be responsible for the reduction of total carbohydrates content in 18MSP bar as compared to the control one. The protein and fat contents observed in the developed 18MSP are higher to those reported in other healthy snack bars enriched in quinoa and corn (Kaur, Ahluwalia, Sachdev, & Kaur, 2018; Figuereido de Sousa et al., 2019), and also in biscuits supplemented with moringa dry leaf (Hedhili et al., 2021). Our findings regarding the enhancement of protein, fat and ash contents in MSP-supplemented snack bars are in agreement with those reported in cakes, pasta and breads where wheat flour was replaced by germinated moringa or moringa seed powder (Bolarinwa, Aruna, & Raji, 2019; Chinma et al., 2014; Coello et al., 2021). 3.3. Content of thiamine and riboflavin As shown in Table 3, 18MSP exhibited a slightly higher thiamine content (983.0 μ g/100 g dm) than the control bar (961.2 μ g/100 g dm), while riboflavin levels increased more than 15-fold after inclusion of MSP in the bar formulation. MSP obtained after germination at 36 ◦C for 96 h exhibits high levels of thiamine and riboflavin (Coello et al., 2020) and their incorporation in snack bars and other processed foods would be an interesting approach to fortify them in B-group vitamins. To our knowledge, there is no information regarding vitamin B 1 and B 2 contents in cereal-based snack bars. However, vitamin content observed in 18MSP bar is higher than that reported in other cereal-based products such as wheat-based bread and pastries made from multigrain flours Table 2 Fracturability scores for snack bar formulations with different solid/liquid ratios (65/35, w/v and 75/25, w/v) and different amount of MSP (0%, 6%, 9%, 12%, 18%, 19% and 26%). Snack bars % MSP Fracturability Control 65/35 0 3.17 ±0.30 a 9MSP 9 3.83 ±0.69 a 18MSP 18 3.00 ±0.58 a 26MSP 26 4.00 ±1.41 a Control 75/25 0 2.83 ±0.36 a 6MSP 6 3.17 ±0.58 a 12MSP 12 3.00 ±1.53 a 19MSP 19 3.20 ±0.98 a Data are the mean ±standard deviation of three replicates. Different lower-case letters indicate statistical differences among samples (p ≤0.05, Duncan test). Table 3 Proximate composition and contents of thiamine, riboflavin and minerals in control and 18MSP snack bars. Control 18MSP Proximate composition (g/100 g dm) Moisture 8.13 ±0.02 b 7.48 ±0.01 a Protein 7.87 ±0.07 a 11.62 ±0.01 b Fat 2.61 ±0.12 a 11.17 ±0.01 b Ash 1.31 ±0.04 a 1.94 ±0.06 b Total carbohydrates 80.06 ±0.01 b 67.72 ±0.01 a Vitamins ( μ g/100 g dm) Thiamine 961.23 ±16.19 a 983.04 ±14.00 b Riboflavin 6.06 ±1.49 a 94.10 ±5.22 b Minerals (mg/kg dm) Calcium (Ca) 480.79 ±12.92 a 883.78 ±3.45 b Magnesium (Mg) 1147.11 ±58.56 a 1536.06 ±17.25 b Potassium (K) 5044.21 ±13.39 a 7298.47 ±117.49 b Sodium (Na) 806.51 ±32.00 a 637.28 ±15.20 b Phosphorus (P) 1818.32 ±60.08 a 2716.31 ±39.49 b Iron (Fe) 25.16 ±1.10 a 32.85 ±1.02 b Zinc (Zn) 19.36 ±0.74 a 22.12 ±1.10 b Manganese (Mn) 14.02 ±1.40 a 13.92 ±1.02 a Copper (Cu) 5.54 ±0 .05 a 6.30 ±0 .13 a Data are the mean ±standard deviation of three replicates. Different lower-case letters indicate statistical differences among samples (p ≤0.05, Duncan test). K.E. Coello et al. LWT 154 (2022) 112828 5 (ˇ Skrov´ ankov´ a & Sikorov´ a, 2010). 3.4. Mineral content 18MSP snack bar contained significantly (p ≤0.05) higher amounts of Ca, Mg, K, P, Fe and Zn and lower levels of Na than control, while no statistical differences (p >0.05) were observed in Mn and Cu contents between both formulations (Table 3). Notable levels of Ca, Mg, K, P and Fe have been found in moringa seeds (Gopalakrishnan, Doriya, & Kumar., 2016; Ijarotimi, Adeoti, & Ariyo, 2013; Olagbemide & Alikwe, 2014), findings that are in agreement with the mineral content observed in 18MSP. The levels of Ca, Mg, K and Zn in 18MSP were notably higher than those reported in other snack bar formulations based in cereal, legume and nuts (Zainal Abidin, Mohd Zin, Abdullah, Rusli, & Zaino, 2020; Momanyi et al., 2020; Silva Lima et al., 2021). It is important to highlight the reduced content of Na in 18MSP, a desirable attribute taking into consideration the current recommendations of World Health Organization regarding the reduction of dietary Na intake to prevent cardiovascular diseases (WHO, 2012). According to the results (Table 3), two servings of 18MSP snack bar (70 g) provide 49% and 35% of the recommended daily intake for Mg and P, respectively (EFSA, 2015). 3.5. Fatty acid composition Table 4 shows the FA profile of control and 18MSP bars. A total of 9 FA were identified in control bar, while 18MSP formulation contained 17 FA, being oleic acid (C18:1n9c) the most abundant, followed by lauric (C12:0) and palmitic (C16:0) acids. These results agree with the FA composition of moringa seed and germinated moringa recently reported (El-Baset Salama, Owon, Osman, Ibrahim, & Matth¨ aus, 2020; Gharsallah, Rezig, Msaada, Chalh, & Soltani, 2021; Gu, Yang, & Wang, 2020). MSP incorporation resulted in the simultaneous increase in the levels of both unsaturated and saturated FA in snack bars. The enhancement of unsaturated FA content in 18MSP bar was due to the increase of monounsaturated FA (MUFA), while a 34% reduction was observed in polyunsaturated FA (PUFA) compared to control. The replacement of oat and quinoa, that are good sources of PUFA (Aparicio-García et al., 2021; Rodríguez G´ omez, Matías Prieto, Cruz Sobrado, & Calvo Magro, 2021), by a moringa seed-derived ingredient containing low PUFA levels (Gu et al., 2020) explains the PUFA reduction in MSP-enriched bar. Unsaturated FA were predominant in 18MSP lipid fraction, representing ~57% of total FA content (49% MUFA and 7% PUFA), while saturated FA accounted for 43% (Table 4). Recent studies have revealed that MUFA intake reduces the levels of serum triglycerides, total cholesterol, low density lipoprotein (LDL)-cholesterol and atherogenic lipoproteins/apolipoproteins, while increase high density lipoprotein (HDL)-cholesterol (DiNicolantonio & O’Keefe, 2018; Ulug & Nergiz-Unal, 2021) and improves microbiota composition (L´ opez-Salazar et al., 2021). Recently, Wiercioch et al. (2018) evidenced that most of commercially available snack bars show an unhealthy fatty acid profile, where saturated FA are more abundant, representing more than 68% of the total FA content. On the other hand, oleic acid exhibits high stability to oxidation (Fuentes de Mendoza et al., 2015), and its presence in snack bars might enhance their shelf-life. Thus, the incorporation of MSP in bar formulation represents a promising strategy to improve the nutritional and healthy properties as well as the shelf-life of snacks currently consumed. 3.6. Amino acid composition The influence of MSP addition on the amino acid profile of snack bars is shown in Table 5. Glu was the main non-essential amino acid (NEAA) in 18MSP bar, followed by Arg, while Leu, Phe and Val were the predominant essential amino acids (EAA). The incorporation of MSP improved about 26% the total amino acid content as compared to control bar. All NEAA, with the exception of Asp and Pro, were significantly (p ≤0.05) enhanced after MSP addition, increasing the total NEAA content by 36% in comparison with the control formulation. Among EAA, a significant increase (p ≤0.05) in His, Val, Met, Ile, Leu, Phe, Tyr and Thr contents was observed in 18MSP prototype, results in line with the amino acid composition reported for moringa seeds (Gu et al., 2020) and with the increase in the total amino acid content (both NEAA and EAA) in moringa seeds after germination (Ijarotimi et al., 2013). However, Cys and Lys levels decreased significantly (p ≤0.05) in 18MSP bars compared to control. The replacement of oat, a good source of both amino acids, by moringa that contains low levels of Cys and in which Lys is the limiting amino acid, can explain the results obtained. Most of EAA often lack in vegetable matrices used for snack elaboration, but MSP contains all of them in a good proportion, pointing out that sprouted Table 4 Fatty acid composition (mg/g dm) in control and 18MSP snack bars. Fatty acids Control 18MSP C6:0 ND 0.16 ±0.01 a C8:0 ND 2.34 ±0.04 a C10:0 ND 2.03 ±0.04 a C12:0 0.24 ±0.01 a 16.98 ±0.28 b C14:0 0.15 ±0.01 a 6.24 ±0.10 b C16:0 4.33 ±0.02 a 9.00 ±0.12 b C16:1n7 ND 0.78 ±0.01 a C18:0 0.55 ±0.02 a 4.43 ±0.07 b C18:1n7c 0.27 ±0.01 a 3.26 ±0.06 b C18:1n9c 10.02 ±0.03 a 49.67 ±0.61 b C18:2n6c 12.48 ±0.01 b 8.22 ±0.05 a C18:3n3 0.72 ±0.03 b 0.53 ±0.01 a C20:0 ND 1.96 ±0 .05 a C20:1n9 0.26 ±0.02 a 1.59 ±0 .04 b C22:0 ND 3.81 ±0 .06 a C22:1n9 ND 0.16 ±0 .01 a C24:0 ND 0.72 ±0 .02 a Total saturated 5.26 ±0.03 a 47.66 ±0.72 b Total unsaturated 23.76 ±0.02 a 64.06 ±0.73 b MUFA 10.56 ±0.02 a 55.30 ±0.70 b PUFA 13.20 ±0.03 a 8.76 ±0.05 b Total 29.02 ±0.02 a 111.72 ±1.44 a Data are the mean ±standard deviation of three replicates. Different lower-case letters indicate statistical differences among samples (p ≤0.05, Duncan test). ND: not detected; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids. Table 5 Amino acid composition (mg/g dm) in control and 18MSP snack bars. Amino acids Control 18MSP FAO/WHO/UNU 2011 1 Non-essential amino acids Asp +Asn 6.00 ±0.17 a 6.00 ±0.05 a Glu +Gln 13.07 ±0.11 a 18.23 ±0.07 b Ser 3.48 ±0.02 a 3.96 ±0.05 b Gly 3.72 ±0.04 a 4.92 ±0.01 b Arg 5.03 ±0.12 a 10.35 ±0.16 b Ala 3.55 ±0.01 a 4.51 ±0.04 b Pro 1.93 ±0.30 a 2.00 ±0.35 a Total NEAA 36.77 ±0.77 a 49.97 ±0.62 b Essential amino acids His 2.43 ±0.03 a 3.06 ±0.02 b 10 Val 3.58 ±0.002 a 4.11 ±0.03 b 26 Met 1.02 ±0.01 a 1.51 ±0.03 b 10 Cys 1.19 ±0.07 b 0.66 ±0.06 a 4 Ile 2.36 ±0.04 a 2.84 ±0.02 b 20 Leu 5.15 ±0.06 a 5.99 ±0.04 b 39 Phe 3.74 ±0.05 a 4.38 ±0.04 b 25 2 Tyr 2.01 ±0.13 a 2.31 ±0.06 b Lys 3.43 ±0 .08 b 3.14 ±0 .05 a 30 Thr 2.42 ±0.09 a 2.86 ±0.09 b 15 Total EAA 27.33 ±0.52 a 28.01 ±0.06 a Total amino acids 64.10 ±1.29 a 80.83 ±0.70 b Data are the mean ±standard deviation of three replicates. Different lower-case letters indicate statistical differences among samples (p ≤0.05, Duncan test). NEAA: non-essential amino acids; EAA: essential amino acids. K.E. Coello et al. LWT 154 (2022) 112828 6 moringa is a good alternative to current plant ingredients used for snack bar manufacturing. 3.7. GABA content GABA content in 18MSP formulation (36.4 mg/100 g dm) was 20% higher than that observed in the control bar (Table 6). During sprouting, endogenous glutamate decarboxylase enzyme is activated, producing GABA from glutamic acid, being moringa sprouted at 36 ◦C for 96 h a good source of this non-protein amino-acid (Coello et al., 2020). In recent years, GABA has aroused increasing interest for both food researchers and technologists that have conducted extensive studies for developing GABA-enriched foods (Cataldo, Elean, Savoy de Giori, Saavedra, & Hebert, 2020; Hong Le, Parmentier, Trung Le, & Raes, 2021) since it shows numerous physiological functions, such as reduction of blood pressure, protection against cancer, enhancement of immunity, induction of relaxation and improvement of brain function (Boonstra et al., 2015; Sahab, Subroto, Balia, & Utama, 2020). The production of snack bars enriched in GABA is an innovative approach to improve well-being and health of consumers. 3.8. Content of total and individual GLS The supplementation of snack bars with MSP significantly (p ≤0.05) increased the content of both individual and total GLS (Table 6). Glucomoringin (4α -rhamnopyranosyloxy-benzyl GLS) was the only GLS present in the control sample and the content of this compound increased more than 148-fold in snack bars after the addition of MSP, representing 96% of total GLS in 18MSP. 18MSP prototype also showed small amounts of glucosinalbin (4-hydroxybenzyl GLS) and glucotropaeolin (benzyl GLS), compounds that were not detected in control bar. As consequence, total GLS increased more than 150-fold in 18MPS bar in comparison with control. In agreement with our results, previous studies have identified glucomoringin as the main GLS in M. oleifera seeds (Amaglo et al., 2010; Chen et al., 2019; Maldini et al., 2014). Small amounts of glucotropaeolin and glucosinalbin have been also detected in moringa seeds by Maldini et al. (2014). The presence of GLS in 18MSP snack bar is of great interest since they are secondary metabolites that are catabolized by the endogenous plant myrosinase enzyme to yield isothiocyanates, with recognized health-promoting properties (Lopez-- Rodriguez, Gayt´ an-Martínez, Reyes-Vega, & Loarca-Pi˜ na, 2020). In fact, GLS contained in moringa have shown chemoprotective potential through cytoprotective, anticarcinogenic and antioxidant effects (Fahey et al., 2018). Therefore, snack bars rich in moringa GLS might provide a wide range of health benefits to consumers. 3.9. Content of soluble phenolic compounds (SPC) Control snack bars showed SPC levels of 267.4 mg GAE/100 g dm, which increased 70.5% in 18MSP bar (Table 6). Germination has shown to play a remarkable role in improving total phenolic content in a wide range of seeds. In moringa, high germination temperatures (32–40 ◦C) efficiently favored the accumulation of phenolic compounds (Coello et al., 2020; Le´ on-L´ opez et al., 2019). Plants activate adaptive strategies in response to abiotic stress such as high temperatures that involve activation of phenylalanine ammonia lyase (PAL), enzyme responsible for the synthesis of phenolics. The activation of endogenous seed cellulases, endoxylanases and esterases during sprouting leads to disruption of cell wall components and the release of bound phenolic acids (Sharma, Singh, & Sing, 2019), that might also contribute to the high SPC levels in MSP and, consequently, in 18MSP snack bars. Since several phenolic compounds exhibit powerful antioxidant activity, the development of snack bars enriched in phenolics is of undoubtedly interest. 3.10. Antioxidant activity As shown in Table 6, 18MSP snack bars displayed a notable antioxidant activity (647.2 mg TE/100 g dm), which was 22% higher than that observed in control bars. These results support recently findings regarding the enhancement of oxygen radical absorbance capacity in sprouted moringa (Coello et al., 2020; Le´ on-L´ opez et al., 2019). The improvement of antioxidant activity in 18MSP bar compared to control can be attributed to the accumulation of compounds with peroxyl-scavenging activity in MSP after germination. Phenolic acids and flavonoids, compounds with recognized antioxidant activity (Gu, Howel, Dunshea, & Suleria, 2019) that have been identified in moringa seed oil (Gharsallah et al., 2021) might contribute to the enhanced ORAC values observed in 18MSP bar compared to control. GABA also shows free-radical scavenging ability (Zhao, Xie, Wang, Gu, & Yang, 2021), and it may act, together with phenolics and vitamins present in MSP, as antioxidant responsible for the enhanced radical scavenging activity observed in 18MSP. 3.11. Sensory analysis Fig. 1 shows the scores given by panelists to sensory attributes of control and 18MSP snack bars. The sensory scores revealed that MSP supplementation affected the consumer perception of the different sensory attributes studied. Texture and odour were the attributes better appreciated in 18MSP bars by the panelists, since they liked extremely to 60–70% of them and received better scores than control bar. Regarding appearance, not significant differences between control and 18MSP bars were observed, results of notable importance since appearance is one of the most important parameters determining the choice of a food product (Ciurzy´ nska et al., 2019). The most pronounced differences between both formulations were observed in taste, attribute which received lower scores in 18MSP formulation than control. Bitterness and astringency are attributes associated with moringa taste, due to the presence of GLS and phenolic compounds, which together with unsaturated FA provide nutty and green sensory notes (Chinma et al., 2014; Methakullawat & Lorjaroenphon, 2019). These attributes could be responsible for the reduction of taste scores in 18MSP bar. Despite the sensory results for taste, it must be taking into account that 18MSP formulation liked moderately or slightly to 60% of the panelists. The addition of higher levels of dry banana and coconut or novel tasty ingredients that mask the unpleasant bitter and astringent taste of germinated moringa or the debittering of sprouted moringa flour (Ogunsina, Radha, & Indrani, 2011) might improve the sensory perception of 18MSP bar. Regarding overall acceptability, 18MSP bar liked to 70% of panelists, while control bar liked to 60%. The improved texture and odour might be responsible for the highest acceptability of 18MSP bar. A previous study performed by our group showed that supplementation of fettuccini with 15–30% of MSP reduced taste scores and overall acceptability (Coello et al., 2021), while Chinma et al. (2014) observed that replacement of wheat by 10%–40% of germinated moringa flour did not change the taste and overall acceptability of the cakes obtained. Table 6 Content of GABA, SPC and antioxidant activity in control and 18MSP snack bars. Control 18MSP GABA 36.40 ±0.84 a 43.33 ±1.09 b Glucomoringin 0.20 ±0.06 a 29.62 ±1.29 b Glucosinalbin ND 1.06 ±0.06 a Glucotropaeolin ND 0.21 ±0.06 a Total GLS 0.20 ±0.06 a 30.88 ±1.36 b SPC 267.39 ±11.84 a 455.97 ±32.29 b Antioxidant activity 531.13 ±51.73 a 647.17 ±48.85 b Data are the mean ±standard deviation of three replicates. Different lower-case letters indicate statistical differences among samples (p ≤0.05, Duncan test). GABA: γ-amino butyric acid; GLS: glucosinolates; ND: not detected; SPC: soluble phenolic compounds. K.E. Coello et al. LWT 154 (2022) 112828 7 3.12. Estimation of capacity of manufacturing snack bars and selling cost Taking into account the mass balance calculation of each stage involved in snack bar manufacturing, the efficiency of the process was 90.3%. Consequently, it was estimated that with 45,000 g per batch/h of raw material mixture it is possible to obtain 1161 bars of 35 g with an estimated production unit (calculated according to Eq. (3)) and selling cost (calculated according Eq. (4)) of 0.45$ and 0.75$, respectively. The analysis of the price of commercial snack bars in Ecuador allowed to conclude that it ranges from 0.36$/unit to 1.14$/unit, with an average price of 0.62$/unit. Taking into account that 18MSP snack bar has a high added-valued due to its notable content of nutrients and healthpromoting compounds and that “healthiness” is one of the main reasons for determining food choices and consumer’s willingness to pay (Timpanaro, Bellia, Foti, & Scuderi, 2020), it can be speculated that 0.75 $ would be an acceptable price for 18MSP snack bar for Ecuadorian consumers. 3.13. Estimation of consumer’s willingness to purchase snack bars supplemented with MSP Before turning to consumers’ willingness to pay estimation, a closer look at snack bar purchasing behavior (Fig. S2; supplementary material) and also, the knowledge about moringa health benefits and willingness to purchase moringa-enriched snack bars (Fig. S3, supplementary material) by Ecuadorian consumers was taken. In total, 33% of responders consume snack bars once a week, 29% consume them twice or more times a week, 20% once a month and 18% twice or three times a month (Fig. S2a). The shelf-reported frequency of consumption of snack bars by Ecuadorian consumers is lower than that observed by Leite Munhoz et al. (2014), who reported a frequency of cereal bar consumption of three times a week in 60% of Brasilian responders. However, lower cereal bar intake was reported by Costa et al. (2021), who found that 17% of Brasilian responders consumed cereal bars 1–4 times a week. The main reason for the consumption of snack bars pointed by Ecuadorian responders was the taste (34%), followed by their nutritional value and energy-providing (25%), prevention of diseases (18%), practicality (12%) and other reasons (11%) (Fig. S2b). Regarding moringa, 52% of responders declared to know its nutritional and health benefits, while 48% were unaware of their favourable effects on health (Fig. S3a). As regards of purchase intention, 79% of the participants indicated that they probably would buy snack bars containing moringa (Fig. S3b), suggesting that they represent an opportunity of innovation in snack bars market niche in Ecuador. Moringa is a widely known tree in Ecuador, where leaves are consumed due to their nutritional and health-related properties (Guaycha-P´ erez, Jaramillo-Jaramillo, Cuenca-Buele, Tocto-Le´ on, & M´ arquez-Hern´ andez, 2017), thus explaining the results obtained in the questionnaire. Referring to the willingness to pay, 69% of responders declared that they were willing to pay between 0.5 and 1$ for the purchase of moringa-enriched snack bars, while 22% were willing to pay this product at higher price of 1.5–2$ (Fig. S3c). Only 8% of consumers were willing to pay 2.5–3$ for this snack bar prototype, while prices of 3.5–4$ were only accepted by 1% of participants. Taking into account that the production costs per unit was estimated in 0.45$ and the selling cost in 0.75$ (section 3.11), a price of 1$ per a MSP-supplemented snack bar Fig. 1. Scores of sensory attributes of control and 18MSP snack bars. Liked extremely. Liked moderately. Liked slightly. Indifference. Disliked slightly. K.E. Coello et al. LWT 154 (2022) 112828 8 can be considered acceptable for both consumers and industry. These results are in agreement with previous studies reporting prices of 1 € (1.18$) for a cereal bar made with brewery spent grains (Stelick, Sogari, Rodolfi, Dando, & Paciulli, 2021). 4. Conclusions The relevance and innovation of the present study lie in the incorporation of moringa sprout powder (MSP) as an ingredient for manufacturing nutritive-dense and healthy snack bars that offer new opportunities for the Ecuadorian snack market. Snack bar formulation containing 18% of MSP (18MSP) was a good source of protein, unsaturated FA, and micronutrients, while its content of carbohydrates was lower than in control bar. MSP incorporation also enhanced the content of bioactive compounds with recognized health promoting properties such as SPC, GABA, total and individual GLS compared to the control formulation. MSP-enriched bar was well accepted by panelists, primarily due to its texture and odour, while its taste received lower scores than the control formulation. Questionnaires performed to potential consumers indicated that most of them would purchase the product at a price of 1$, an acceptable price for both consumers and industry. The findings of this study demonstrated that MSP might represent a valuable ingredient to improve nutritional and health-promoting properties of snack bars. Further studies will be performed in order to obtain more information on the composition of these snacks with the aim to commercialize them. CRediT authorship contribution statement Karín E. Coello: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft. Juana Frias: Conceptualization, Methodology, Validation, Investigation, Resources, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition. Cristina Martínez-Villaluenga: Methodology, Validation, Investigation, Resources, Writing – review & editing. María Elena Cartea: Methodology, Validation, Investigation, Resources, Writing – review & editing. Pablo Velasco: Methodology, Validation, Investigation, Writing – review & editing. Elena Pe˜ nas: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition. Declaration of competing interest None. Acknowledgments This study was funded by grants AGL2015-67598-R (MINECO/ FEDER), AGL2017-83718-R (AEI/FEDER, UE), and intramural project grant number PI201870I097 (CSIC). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.lwt.2021.112828. References Al Juhaimi, F., Ghafoor, K., Babiker, E., Mattha¨ aus, B., & ¨ Ozcan, M. M. (2017). The biochemical composition of the leaves and sedes meals of moringa species as nonconventional sources of nutrients. Journal of Food Biochemistry, 41, Article e12322. https://doi.org/10.1111/jfbc.12322 Aluko, O., Brai, M. R., & Adelore, A. O. (2013). Evaluation of sensory attributes of snack from maize-moringa seed flour blends. International Journal of Nutrition and Food Engineering, 7, 944–946. https://doi.org/10.5281/zenodo.1088095 Alvarez, M. D., Fuentes, R., Guerrero, G., & Canet, W. (2017). 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